Circulating Tumor DNA Assay Detects Merkel Cell Carcinoma Recurrence, Disease Progression, and Minimal Residual Disease: Surveillance and Prognostic Implications

PURPOSE Merkel cell carcinoma (MCC) is an aggressive skin cancer with a 40% recurrence rate, lacking effective prognostic biomarkers and surveillance methods. This prospective, multicenter, observational study aimed to evaluate circulating tumor DNA (ctDNA) as a biomarker for detecting MCC recurrence. METHODS Plasma samples, clinical data, and imaging results were collected from 319 patients. A tumor-informed ctDNA assay was used for analysis. Patients were divided into discovery (167 patients) and validation (152 patients) cohorts. Diagnostic performance, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), was assessed. RESULTS ctDNA showed high sensitivity, 95% (discovery; 95% CI, 87 to 99) and 94% (validation; 95% CI, 85 to 98), for detecting disease at enrollment, with corresponding specificities of 90% (95% CI, 82 to 95) and 86% (95% CI, 77 to 93). A positive ctDNA during surveillance indicated increased recurrence risk, with hazard ratios (HRs) of 6.8 (discovery; 95% CI, 2.9 to 16) and 20 (validation; 95% CI, 8.3 to 50). The PPV for clinical recurrence at 1 year after a positive ctDNA test was 69% (discovery; 95% CI, 32 to 91) and 94% (validation; 95% CI, 71 to 100), respectively. The NPV at 135 days after a negative ctDNA test was 94% (discovery; 95% CI, 90 to 97) and 93% (validation; 95% CI, 89 to 97), respectively. Patients positive for ctDNA within 4 months after treatment had higher rates of recurrence, with 1-year rates of 74% versus 21% (adjusted HR, 7.4 [95% CI, 2.7 to 20]). CONCLUSION ctDNA testing exhibited high prognostic accuracy in detecting MCC recurrence, suggesting its potential to reduce frequent surveillance imaging. ctDNA also identifies high-risk patients who need more frequent imaging and may be best suited for adjuvant therapy trials.


INTRODUCTION
Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer, associated with high mortality and a 40% recurrence rate within 5 years. 1 To monitor for potential recurrence, patients are typically subjected to serial fullbody imaging using computed tomography (CT) or positron emission tomography (PET)-CT for up to 5 years. 2,3 the majority of MCC cases are caused by clonal integration of the Merkel cell polyomavirus (MCPyV) into the tumor genome, the MCPyV oncoprotein antibody test is a serology assay that has been used as a tumor marker. 4However, only approximately 50% of patients produce MCPyV oncoprotein antibody at the time of disease presence, antibody titers fall slowly over months after the disease is removed, and titers are less reliable after the first recurrence. 5,6Therefore, there is a need for an effective, blood-based biomarker of MCC disease that can be used to stratify patients with high-risk MCC, regardless of viral status.
[18] In this prospective, multicenter, observational study, we assessed the utility of tumor-informed ctDNA for the detection of disease in patients with MCC.We evaluated ctDNA levels at the time of presentation and after initial treatment to determine whether ctDNA could detect clinically evident disease and identify high-risk patients who are likely to have a recurrence.

Study Design and Population
This study was designed as a prospective, multicenter, and observational study of stage I-IV patients with histologically confirmed MCC with a discovery cohort and a validation cohort.All patients provided written informed consent approved by the institutional review board (IRB) at each participating center, and a data-sharing agreement was procured between the institutions.Patients were enrolled between April 2020 and August 2022, and the data cutoff dates were July 8, 2022, and August 31, 2022, for discovery and validation cohorts, respectively.Patients were eligible for enrollment at any time during their disease course, including before or after treatment.Blood samples were collected for ctDNA testing at the time of enrollment, and every 3 months during the surveillance period.Imaging studies, including CT, PET-CT, magnetic resonance imaging, or ultrasound, were obtained at enrollment for primary tumors and per National Comprehensive Cancer Network guidelines for patients in surveillance.If there was an unexpected rise in ctDNA, an additional ctDNA test was performed coupled with an imaging study within 4 weeks (protocol flowchart shown in Appendix Fig A1, online only).Clinical details, including follow-up, disease status at the time of enrollment, recurrences, and imaging results, were collected.Clinically evident disease was defined as MCC that was detected through physical examination, imaging studies, or tissue biopsy.Patients with incomplete clinical data, unattainable ctDNA assay development because of insufficient tissue, or sequencing failure because of comorbid hematologic malignancy or transplant were excluded.Inclusion and exclusion criteria for subgroup analyses are described in each objective in the section below.

ctDNA Assay Using Multiplex Polymerase Chain Reaction-Based Next-Generation Sequencing
Tumor-informed ctDNA assays were designed for each patient as previously described. 11,19The ctDNA assay was centralized and conducted by Natera Inc, the developer and provider of the Signatera assay.Briefly, up to 16 patientspecific, somatic single-nucleotide variants (SNVs) were selected by performing whole-exome sequencing on formalin-fixed paraffin-embedded tumor tissue and matched normal blood samples.The multiplex polymerase chain reaction primers targeting the selected SNVs were used to detect ctDNA in patients' plasma samples.0][21][22][23][24]

Discovery and Validation Cohorts
Patients enrolled by Stanford University and University of Washington were designated as the discovery cohort and patients enrolled by Dana-Farber Cancer Institute, Northwestern University, University of California San Francisco, and Moffitt Cancer Center were designated as the validation cohort.Enrollment and data collection ran from April 2020 through August 2022 in the discovery cohort and from February 2021 through July 2022 in the validation cohort.
A total of 336 patients were initially enrolled across six study sites.Among them, 17 patients were excluded as shown in Figure 1.The remaining 319 patients were included in the analysis, with 167 in the discovery cohort and 152 in the validation cohort.Patient characteristics for the discovery and validation cohorts are summarized in Appendix Table A1 and the Data Supplement (Methods, online only).A total of 562 ctDNA tests were performed over a median follow-up of 295 days in the discovery cohort (median [IQR], 3 [2-5] tests per patient) and 640 ctDNA tests were performed over a median follow-up of 284 days in the validation cohort (median [IQR], 3 [2-4]  recurrence stratified by ctDNA status on the basis of serial ctDNA testing during surveillance; and (3) positive predictive value (PPV) and negative predictive value (NPV) of the ctDNA test for predicting clinical recurrence at each time point during surveillance.Secondary analyses included (1) correlating quantitative ctDNA level with primary tumor size; (2) quantifying risk of recurrence at varying levels of ctDNA positivity, and (3) evaluating whether the detection of ctDNA after completing initial treatment can predict recurrence and risk stratify patients.

Statistical Analysis
The Reporting Recommendations for Tumor Marker Prognostic Studies guidelines were followed in the analysis and reporting of results (Appendix Table A2). 25A detailed statistical analysis plan is included in the Data Supplement.All P values were two-sided and statistical significance was defined as P < .05without adjustment for multiple testing.
The primary analysis plan was developed using the discovery cohort before any statistical analysis of the validation cohort.The study protocol and procedures were not altered over the study period on the basis of any data analysis results.Primary end point analyses were performed on each cohort separately.These primary analyses were performed first on the discovery cohort while developing the analysis plan and determining the ctDNA MTM/mL threshold for detection of MCC, and second on the validation cohort as a prespecified analysis to achieve unbiased estimates of ctDNA performance for the detection of MCC.Secondary end point analyses were conducted using both the discovery and validation cohorts combined as a single cohort to maximize the available sample size.The division of end points into primary and secondary was done on the basis of the results from the discovery cohort, before any analysis of the validation cohort.

Primary End Point Analyses
The sensitivity and specificity of the ctDNA test for detecting clinically evident disease at enrollment were estimated using the first ctDNA test for all patients with determinable clinical disease status.The performance of the ctDNA test at different thresholds was also considered using receiver operating characteristic (ROC) curve analysis and summarized using the AUC.The ROC analysis of ctDNA in the discovery cohort was used to determine the ctDNA threshold for positivity to be assessed in the validation cohort.In the surveillance setting, the association of ctDNA status with risk of recurrence during serial testing was performed by stratifying patients as ctDNA-positive at any time point versus patients who remained ctDNA-negative, where ctDNA status was treated as a time-varying covariate to account for immortal time bias. 26Cox regression models were used to compare recurrence risk between positive and negative groups while adjusting for other risk factors.The PPV and NPV of ctDNA status for recurrence during surveillance were estimated at multiple intervals after a positive or negative test using the cumulative incidence function estimator.The unit of analysis for PPV and NPV was the ctDNA test, and the time to recurrence after each test was defined as the time from that test to clinical detection of recurrence, censored at the last follow-up.Clustered bootstrapping was used to account for the nonindependence of multiple tests per patient. 27

Secondary End Point Analyses
Statistical methods for the secondary end point analyses are provided in the Data Supplement.

IRB
All studies were performed in accordance with the Declaration of Helsinki and were approved by the IRB (protocol code Stanford IRB 61461, University of Washington/Fred Hutch Cancer IRB 6585, Dana-Farber/Harvard Cancer Center IRB 09-156, Northwestern University IRB STU00216228, University of California San Francisco IRB 21-35252, and Moffitt Cancer Center IRB 00000971).All patients included in this study provided informed consent for their clinical data to be analyzed for research purposes.
Higher thresholds for ctDNA positivity were also considered using ROC analysis (Appendix Fig A4).Overall, the ctDNA level at enrollment had an AUC of 0.95 (95% CI, 0.92 to 0.99) for discriminating between clinically evident disease and no clinically evident disease in the discovery cohort.Sensitivity dropped rapidly as the ctDNA threshold was increased from 0.00 MTM/mL with minimal improvement in specificity (ctDNA >1 MTM/mL had a sensitivity and a specificity of 80% [53/66] and 93% [89/96], respectively).To avoid this disproportionate loss of sensitivity, the threshold for ctDNA positivity of ctDNA >0.00 MTM/mL was used for validation in the validation cohort.
In the combined cohort, we also explored whether diagnostic performance differed by whether the patient was on immunotherapy at enrollment (n 5 48) or not (n 5 264).Both sensitivity (95% v 94%; P > .99)and specificity (81% v 89%; P 5 .33)were similar in patients on immunotherapy versus not on immunotherapy (Appendix Table A3).Additionally, ctDNA level and primary tumor diameter were significantly correlated (Spearman's r 5 0.83; P < .001;

DISCUSSION
In this multicenter prospective observational study of patients with stage I-IV MCC, we formally validated the utility of a tumor-informed ctDNA assay.This assay may be particularly impactful in the surveillance of patients with this highly lethal malignancy characterized by a high recurrence rate of 40% within 5 years. 1 We demonstrated that the ctDNA assay had high sensitivity (94% in the validation cohort; Fig 2B) in detecting clinically evident disease at enrollment.Additionally, analyses of patients followed during surveillance revealed that a negative ctDNA had a very high NPV (Fig 3) and that a positive ctDNA after curative-intent treatment predicted patients with a high risk of recurrence (Fig 5).
Previous attempts to find accurate and universally effective tumor markers in MCC have fallen short.Detectable antibodies against the MCPyV are present in only 52% of patients with MCC, 5,6 although MCPyV drives up to 80% of MCC tumors.It is a valuable biomarker in antibody-positive patients, with a PPV of 66% for clinically evident recurrence and an excellent NPV of 97% for a decreasing titer.However, its poor sensitivity in the overall MCC population limits its clinical application.
National guidelines recommend surveillance imaging for high-risk patients and as clinically indicated for others but do not specify an interval in either population. 3The results of our study support using ctDNA to guide imaging frequency in patients under surveillance after treatment of MCC. 12 months, the recurrence-free probability was 9% among patients with a positive ctDNA at any time during surveillance, compared with 91% for patients who remained ctDNA-negative (Fig 3A surveillance. 28The HR was 0.58 (95% CI, 0.30 to 1.12; P 5 .10),but the difference was not significant.As adjuvant therapy is more justified in higher-risk patients, using ctDNA positivity after curative-intent therapy for patient selection in future studies may increase the proportion of patients who could benefit from the adjuvant therapy and increase statistical power to detect differences between treated and untreated groups.
Although our primary analysis evaluated the performance of ctDNA positivity, we also explored whether the likelihood of clinically detecting a recurrence at the time of a positive test was related to the quantitative ctDNA level.Our results show that ctDNA can identify minimal residual disease after primary treatment of MCC.The probability of clinical recurrence increased significantly with higher ctDNA levels (AUC, 0.86 [95% CI, 0.80 to 0.92]; Fig 4B , although the recurrence risk was still appreciable even when ctDNA levels were relatively low (17% when positive ctDNA <1 MTM/mL).This finding suggests that even low ctDNA levels should have clinical follow-up, although the ctDNA value may guide the urgency and frequency of follow-up.Further study is needed to develop more formal guidelines on interpreting the quantitative levels.This study has several limitations.Our patient population sought treatment at tertiary care centers across the United States and thus may not be representative of all patients with MCC.There were real-world variations in primary treatment modalities and follow-up intervals for physical examinations, imaging, and ctDNA collection.Sensitivity and specificity of ctDNA were calculated on the basis of the clinical disease status assessed at the time of the first blood draw for ctDNA.This methodology does not account for new clinically evident disease subsequently found during follow-up.PPV and NPV were determined on the basis of follow-up examinations but could be affected by variation in follow-up intervals.Although our median follow-up is only 295 days, the high recurrence rate of MCC allowed for sufficient statistical power.
In summary, tumor-informed ctDNA testing is a highly accurate and prognostic biomarker for surveillance of patients with MCC, identifying low-risk patients who do not require frequent imaging, and identifying high-risk patients who require more frequent imaging.This assay can aid in early detection of recurrent or metastatic disease, although further studies with longer follow-up are needed to assess the impact on disease-specific survival.Future studies should evaluate the utility of this assay for identifying patients who are most likely to benefit from adjuvant therapy, and for monitoring tumor response to immunotherapy.Finally, as the utilization of this ctDNA assay may decrease the need for imaging follow-up in patients with undetectable levels or on systemic therapies, future studies should additionally assess the impact on the cost of care.
Figure 1.The remaining 319 patients were included in the analysis, with 167 in the discovery cohort and 152 in the validation cohort.Patient characteristics for the discovery and validation cohorts are summarized in Appendix Table A1 and the Data Supplement (Methods, online only).A total of 562 ctDNA tests were performed over a median follow-up of 295 days in the discovery cohort (median [IQR], 3 [2-5] tests per patient) and 640 ctDNA tests were performed over a median follow-up of 284 days in the validation cohort (median [IQR], 3 [2-4] tests per patient).Swimmer plots of all patients in the discovery cohort (Appendix Fig A2) and the validation cohort (Appendix Fig A3) showing individuallevel data on stage, disease status, treatments, ctDNA tests, scans, and outcomes over time are included in the Data Supplement.
Fig 2C) among the 20 patients enrolled before initial treatment with local disease only and detectable ctDNA.
Risk of Recurrence Stratified by ctDNA Status During SurveillanceDuring surveillance, 119 patients (369 plasma samples) from the discovery cohort and 96 patients (288 plasma samples) from the validation cohort underwent serial ctDNA testing (Fig1).These were stage I-IV patients who clinically had no evidence of disease at the start of surveillance.The median interval between ctDNA tests was 91 days (IQR, 77-107) in the discovery cohort and 83 days (IQR, 56-98) in the

FIG 4 .
FIG 4. Likelihood of clinical detection of recurrence at different quantitative levels of ctDNA.(A) ctDNA levels from positive tests, stratified by whether the positive test was within 90 days of a clinical recurrence.Units are MTM per mL.Positive ctDNA levels drawn within 90 days of a recurrence (n 5 79 tests) were significantly higher than levels drawn >90 days before a recurrence (n 5 67 tests; P < .001).(B) Estimated likelihood of a recurrence being clinically detectable within 90 days before or after a positive ctDNA test, stratified at different ctDNA levels.Gray bars show risk of clinical recurrence when the ctDNA level was at or higher than the given threshold on the x-axis and the white bars show the risk of clinical recurrence when the ctDNA level was positive but below the given threshold.ctDNA, circulating tumor DNA; MTM, mean tumor molecule.
IQR, 0.7-16 MTM/mL; range, 0.03-4490 MTM/mL) in stage I-II patients with detectable ctDNA, local disease only, and enrolled before initial treatment (n 5 20; Spearman's r 5 0.83; P < .001).CED, clinically evident disease; ctDNA, circulating tumor DNA; MCC, Merkel cell carcinoma; MTM, mean tumor molecule.a Specificity was based on the absence of clinically evident disease at the time of enrollment, without consideration of subsequent recurrences.Among these, 24 patients in the discovery cohort recurred and two died over a median follow-up of 267 days.Comparatively, 25 patients in the validation cohort recurred with no deaths over a median follow-up of 194 days.The risk of recurrence was significantly higher in patients who were ctDNA-positive at any point during surveillance compared with those who remained ctDNA-negative in both the discovery cohort (hazard ratio [HR], 6.8 [95% CI, 2. CFIG 2. Diagnostic performance of ctDNA at enrollment for MCC disease status in the discovery and validation cohorts.(A) Flowchart for sensitivity and specificity calculations on the basis of disease status and ctDNA status at enrollment.(B) Diagnostic accuracy of ctDNA at enrollment.The sensitivity of ctDNA for CED, defined as detection of MCC on imaging or physical examination, at enrollment was 63/66 (95%; 95% CI, 87 to 99) in the discovery cohort and 59/63 (94%; 95% CI, 84 to 99) in the validation cohort.The specificity of ctDNA at enrollment was 86/96 (90%; 95% CI, 82 to 95) in the discovery cohort and 75/87 (86%; 95% CI, 77 to 93) in the validation cohort.(C) Relationship of primary tumor size (median, 1.8 cm; IQR, 1.0-2.2cm; range, 0.4-12 cm) and the corresponding ctDNA level at enrollment (median, 4.2 [MTM/mL; Journal of Clinical Oncology ascopubs.org/journal/jco| Volume 42, Issue 26 | 3155 ctDNA in MCC: Recurrence and Prognosis validation cohort.estimated risk of clinical recurrence detection within 90 days was 69% (72/105) when ctDNA was above 1 MTM/ mL and 17% (7/41) when ctDNA was below 1 MTM/mL.The estimated risk was 100% (23/23) when ctDNA was above 100 MTM/mL and 46% (56/123) when ctDNA was positive and below 100 MTM/mL (Fig 4B).
(Continued).follow-up are shown using orange circles (positive test) and blue circles (negative test).Surveillance periods, between when a patient was determined clinically to be negative for disease and a recurrence or end of follow-up, are indicated by the green lines.Recurrences or disease progression (X), death (upside-down triangle), and other relevant treatments and procedures are also depicted.AJCC, American Joint Committee on Cancer; ctDNA, circulating tumor DNA; MCC, Merkel cell carcinoma.HRs relating ctDNA status and other risk factors with recurrence during surveillance.HRs are adjusted for all other factors shown.Patients who were ctDNA-positive at any point during surveillance compared with those who remained ctDNA-negative in both the discovery cohort (HR, 7.0 [95% CI, 2.6 to 18.7]; P < .001)and the validation cohort (HR, 19 [95% CI, 7.1 to 51]; P < .001)afteraccountingforotherrisk factors.Detailed numeric results are shown in Appendix Table A4.CI, confidence interval; ctDNA, circulating tumor DNA; HR, hazard ratio.State the method of case selection, including whether prospective or retrospective and whether stratification or matching (eg, by stage of disease or age) was used.Specify the time period from which cases were taken, the end of the follow-up period, and the median follow-up time Give rationale for sample size; if the study was designed to detect a specified effect size, give the target power and effect size A2-3 Statistical analysis methods 10Specify all statistical methods, including details of any variable selection procedures and other model-building issues, how model assumptions were verified, and how missing data were handled Describe the flow of patients through the study, including the number of patients included in each stage of the analysis (a diagram may be helpful) and reasons for dropout.Specifically, both overall and for each subgroup extensively examined report the numbers of patients and the number of events Present univariable analysis showing the relation between the marker and outcome, with the estimated effect (eg, hazard ratio and survival probability).Preferably provide similar analyses for all other variables being analyzed.For the effect of a tumor marker on a time-to-event outcome, a Kaplan-Meier plot is recommendedM8-9, F3, F5, ST416For key multivariable analyses, report estimated effects (eg, hazard ratio) with CIs for the marker and, at least for the final model, all other variables in the model F5, ST4 17 Among reported results, provide estimated effects with CIs from an analysis in which the marker and standard prognostic variables are included, regardless of their statistical significance Abbreviation: REMARK, Reporting Recommendations for Tumor Marker Prognostic Studies.a The following prefixes were used for page numbers and numbers of figures and tables: M 5 main text (starting at introduction section); A 5 appendix; F 5 figure in main text; T 5 table in main text; SF 5 supplemental figure; ST 5 supplemental table.Journal of Clinical Oncology ascopubs.org/journal/jco| Volume 42, Issue 26 ctDNA in MCC: Recurrence and Prognosis